DESIGNING A SHUTTLE VECTOR FOR PROTEIN PRODUCTION IN PICHIA PASTORIS - PowerPoint PPT Presentation

DESIGNING A SHUTTLE VECTOR FOR PROTEIN PRODUCTION IN PICHIA PASTORIS pastoris i PICHIA Georgia State University

School Background  Georgia State University was founded as an extension of the Georgia School of Technology‟s “Evening School of Commerce” back in 1913  It has now become the second largest research institution in the University System of Georgia  GSU „s has a 34 acre campus in the heart of Downtown Atlanta  This year GSU got a record high enrollment of above 30,000 students  This is GSU‟s second year participating in iGEM . Petite. H Parker Science Building

Introduction  Historically two organisms have been most commonly used as hosts for recombinant protein production  Escherichia coli Saccharomyces cerevisiae  Limitations of E. coli  Limitations of S. cerevisiae  No post – translational  Hyperglycosylation modifications  Low yields

Advantages of Using Pichia pastoris  Inexpensive to culture  High production of foreign proteins  Post-translational modification capacity  Strongly inducible promoters  AOXI and AOXII

Promoter  Alcohol Oxidase I promoter  Strongly inducible promoter  Activated by methanol  Inactivated in the presence of glucose  Controls expression of Alcohol Oxidase Methanol Formaldehyde + Hydrogen Peroxide

Primer Design  Forward primer Eco RI Not I Xba I 5‟ 3‟  Reverse primer Pst I Not I Spe I 5‟ 3‟  Isolated Gene Eco I Not I Xba I Spe I Not I Pst I

Isolation of Alcohol Oxidase I  Alcohol Oxidase I promoter gene is 940bp  Addition of the restriction sites using primers should add 40bp  As seen in the figure a band was present slightly below 1000bp indicating that AOX1 was successfully isolated.

Vector Design  Alcohol Oxidase I Promoter Rep pMB1origin BBa_K165058

Vector Design Rep pMB1 origin BspH1 EarI Zeocin Resistance

Rep pMB1 origin Vector Design

Homologous Recombination Integration of the vector into the chromosome of P . pastoris in a single cross over event

Future Applications  Characterize the Alcohol Oxidase promoter  Fluorescence of the yeast  Swap with Pichia promoters  Example  Gly-3-Phosphate Dehydrogenase  Formaldehyde Dehydrogenase  Transcription Elongation Factor  Production of eukaryotic proteins and production of vaccines

2011 Team Accomplishments  Established GSU Synthetic Biology Club  Created 3-semester iGEM course for future development  Received $45,000 departmental funding for lab equipment  Hosted multiple fundraising events  Kaplan Auction  Bake Sales  Keg Parties  Racquetball Tournament

Acknowledgements  Dr. Sidney Crow  Sarah Boyd  Dr. George Pierce  Chris Cornelison  Dr. Malcolm Zellars  GSU Synthetic Biology Club  Dr. Eric Gilbert  KAPLAN  Dr. Paul Ulrich  Anatolia Café